This application is a national phase application under 35 U.S.C. 371 of PCT/CA97/00980 filed Dec. 19, 1997, which is a continuation-in-part of U.S. patent application Ser. No. 08/770,850 filed Dec. 20, 1996 (now U.S. Pat. No. 6,082,820).
The present invention relates to biodegradable microparticles for delivery of a biologically active material and is particularly concerned with such microparticles that are targetable to particular cell types.
Vaccines have been used for many years to protect humans and animals against a wide variety of infectious diseases. Such conventional vaccines consist of attenuated pathogens (for example, polio virus), killed pathogens (for example, Bordetella pertussis) or immunogenic components of the pathogen (for example, diphtheria toxoid and hepatitis B surface antigen).
Some antigens are highly immunogenic and are capable alone of eliciting protective immune responses. Other antigens, however, fail to induce a protective immune response or induce only a weak immune response. The immune response of a weakly immunogenic antigen can be significantly enhanced if the antigens are co-administered with adjuvants. Adjuvants enhance the immunogenicity of an antigen but are not necessarily immunogenic themselves. Adjuvants may act by retaining the antigen locally near the site of administration to produce a depot effect facilitating a slow, sustained release of antigen to cells of the immune system. Adjuvants can also attract cells of the immune system to an antigen depot and stimulate such cells to elicit immune responses. Adjuvants have been identified that enhance the immune response to antigens delivered parenterally.
Adjuvants are commonly employed with antigen in vaccine formulations whereby the induction of systemic immunity through parenteral immunization (intramuscular or subcutaneous) is obtained. This approach is suitable for infectious agents gaining access to the body via damaged skin (i.e. Tetanus), however, there are problems encountered due to side-effects and associated toxicity of many adjuvants administered in this fashion. Only those vaccines formulated from aluminum salts (aluminum phosphate or aluminum hydroxide) find routine use in human and veterinary vaccination. However, even these adjuvants are not suitable for use with all antigens and can also cause irritation at the site of injection. There is a clear need to develop adjuvants which safely enhance the immunogenicity of antigens at the site of injection.
There are other problems specific to the nature of the antigen being used. For example, most conventional non-living vaccines require multiple doses for effective immunization. Live attenuated vaccines and many nonliving liquid vaccines suffer from the need for controlled storage conditions and are susceptible to inactivation (e.g. thermal sensitivity). There are also problems associated with combining vaccines in single dosage forms, due to adjuvant incompatibilities, pH, buffer type and the presence of salts.
Mucosal immunity is induced primarily by induction of secretory immunoglobulin (sIgA) in intestinal, bronchial or nasal washings and other external secretions. For example, parenteral cholera vaccines have been shown to offer limited protection whereas the more recently developed oral form is highly effective (ref. 1xe2x80x94throughout this specification, various references are referred to in parenthesis to more fully describe the state of the art to which this invention pertains. Full bibliographic information for each citation is found at the end of the specification, immediately preceding the claims. The disclosures of these references are hereby incorporated by reference into the present disclosure). Studies with human volunteers have shown that oral administration of influenza vaccine is effective at inducing secretory anti-influenza antibodies in nasal secretions and substances have been identified which might be useful as adjuvants for such ingested vaccines. However, most of these adjuvants are relatively poor in terms of improving immune responses to ingested antigens. Currently, most of these adjuvants have been determined to be safe and efficacious in enhancing immune responses in humans and animals to antigens that are administered via the orogastrointestinal, nasopharyngeal-respiratory and genital tracts or in the ocular orbits. However, administration of antigens via these routes is generally ineffective in eliciting an immune response. Although the above example illustrates the potential of these immunization modes, the development of vaccine formulations for use by these routes has been slow for various reasons. The inability to immunize at the mucosal surface is generally believed to be due to include:
(i) antigen degradation via the acid and/or proteolytic enzymes present during the transit to the mucosal surfaces;
(ii) antigen degradation by secretions presented at the mucosal epithelium;
(iii) limited adsorption across the mucosal epithelium;
(iv) the dilution of the antigen to a concentration that is below that required to induce immune responses; and
(v) ineffective adjuvants and/or delivery systems.
The problems associated with the use of adjuvants in parenteral vaccine formulations and the lack of suitable systems for vaccine delivery to mucosal sites understates the need for new techniques that are effective when administered by various routes and are inherently free from associated toxicity concerns or side-effects.
It is also desired to provide vaccine delivery in a single dosage form for both human and animal immunizations as this has the advantage of reducing time and cost, and in human medicine, increases patient compliance which is of extreme importance in developing countries where access is restricted. This is especially true for infants within these countries.
In order to increase immune responses to administered antigens, a carrier may be used to protect the antigen from degradation and also modulate the uptake of these materials in vivo. Sensitive antigens may be entrapped to protect them against destruction, reduction in immunogenicity or dilution. Methods for formulating a carrier include dispersing an antigen within a polymeric matrix (monolithic matrix) or by the coating of a polymeric material around an antigen to give an outer protective wall (core-shell). The manipulation of the formulation protocol can allow for control over the average size of these materials. This has been shown to be important for the uptake of particulates via oral delivery at specialized M-cells of the Peyers patches within the intestinal tract.
U.S. Pat. No. 5,151,264 describes a particulate carrier of a phospholipid/glycolipid/polysaccharide nature that has been termed Bio Vecteurs Supra Moleculairs (BVSM). The particulate carriers are intended to transport a variety of molecules having biological activity in one of the layers thereof. However, U.S. Pat. No. 5,151,264 does not describe particulate carriers containing antigens for immunization and particularly does not describe particulate carriers for immunization via the orogastrointestinal, nasapharyngeal-respiratory and urogenital tracts and in the ocular orbits or other mucosal sites.
Eldridge et al.(refs 2 and 3) observed the delayed release of antigen in vivo from biodegradable microspheres manufactured from polylactide-co-glycolide copolymer also known as PLG or PLGA. Numerous other polymers have been used to encapsulate antigens for formulation into microparticles and some of these include polyglycolide, polylactide, polycaprolactone, polyanhydrides, polyorthoesters and poly(xcex1-hydroxybutyric acid).
U.S. Pat. No. 5,075,109 describes encapsulation of the antigens trinitrophenylated keyhole limpet hemocyanin and staphylococcal enterotoxin B in 50:50 poly (DL-lactide-co-glycolide). Other polymers for encapsulation are suggested, such as poly(glycolide), poly(DL-lactide-co-glycolide), copolyoxalates, polycaprolactone, poly(lactide-co-caprolactone), poly(esteramides), polyorthoesters and poly(xcex1-hydroxybutyric acid), and poly anhydrides. The encapsulated antigen was administered to mice via gastric intubation and resulted in the appearance of significant antigen-specific IgA antibodies in saliva and gut secretions and in sera. As is stated in this patent, in contrast, the oral administration of the same amount of unencapsulated antigen was ineffective at inducing specific antibodies of any isotype in any of the fluids tested. Poly(DL-lactide-co-glycolide) microcapsules were also used to administer antigen by parenteral injection.
Published PCT application WO 91/06282 describes a delivery vehicle comprising a plurality of bioadhesive microspheres and antigenic vaccine ingredients. The microspheres being of starch, gelatin, dextran, collagen or albumin. This delivery vehicle is particularly intended for the uptake of vaccine across the nasal mucosa. The delivery vehicle may additionally contain an absorption enhancer. The antigens are typically encapsulated within protective polymeric materials.
U.S. Pat. No. 5,571,531 describes particulate carriers comprising a solid matrix of a polysaccharide and a proteinaceous material. A functionalized silicone polymer is bonded to the matrix for the delivery of materials having biological activity.
Although time-delayed release of antigen was shown in the above work, difficulties were encountered when microparticles are manufactured by the described methods. The exposure of biological materials to the organic solvents and physical forces used can lead to denaturation. It may be also be difficult to scale-up the procedures. Furthermore, hydrophilic antigens may be inefficiently encapsulated.
It would be desirable to provide improved carriers without such limitations. It would be particularly desirable to provide polymeric materials which can be formulated into microparticles and microspheres and which contain targeting moieties to target the antigen to preselected ligands. This would have tremendous potential for cells of the immune system.
The present invention is directed towards the production of a novel and useful polymer that has properties suitable for manufacturing by various processes into microparticles and microspheres. In this invention, modifications of existing processing procedures results in significant improvement in encapsulation efficiencies.
This invention is further directed to the production of useful vaccine delivery systems for antigen(s) or antigen and co-adjuvant cocktails by various immunization routes which include parenteral, oral and intranasal.
In accordance with a first aspect of the invention, there is provided a novel biodegradable, biocompatible polymer, including, those having a molecular weight of about 5,000 to about 40,000 daltons, having a backbone of the general formula: 
wherein;
R1, R2, R3, R4 and R5 are selected independently and are selected from H, linear or branched alkyl groups;
R6 is selected from H, an amine protecting group, a spacer molecule or a biologically active species;
X is selected from an O or S group; and
x and y are integers, including values such that at least about 95% of the polymer is comprised of xcex1-hydroxy acid residues.
The novel polymers are derived by copolymerization of monomers comprising at least one xcex1-hydroxy acid or derivative thereof, including cyclic divesters and at least one pseudo-xcex1-amino acid. The xcex1-hydroxy acids are generally of the formula R1R2COHCO2H, where the R1 and R2 groups are H, linear or branched alkyl groups. The xcex1-hydroxy acids may comprise a mixture of xcex1-hydroxy acids, at least one of the mixture of xcex1-hydroxy acids having R1 and R2 groups which are hydrogen and another xcex1-hydroxy acid having an R1 group which is CH3 and R2 which is H. The pseudo-xcex1-amino acids are generally of the formula R5CHNHR6CO2H, where the R5 group is a hydroxyl methyl or methyl thiol group and R6 is an amine protecting group.
The amine protecting groups may be carbobenzyloxy, benzyl, paramethoxybenzyl, benzyloxymethoxy, tert-butyloxycarbonyl or [9-fluorenylmethyloxy]carbonyl.
The xcex1-hydroxy acids are generally selected from L-lactic acid, D,L-lactic acid, glycolic acid, hydroxy valeric acid and hydroxybutyric acid. The at least one pseudo-xcex1-amino acid may be serine.
In a preferred aspect of the invention, the polymers are poly-D,L-lactide-co-glycolide-co-pseudo-Z-serine ester (PLGpZS) and poly-P,L-lactide-co-glycolide-co-pseudo-serine ester (PLGpS).
The polymers may contain biologically active moieties, such as cell bioadhesion groups, macrophage stimulators, polyethylene glycol, poly amino acids and/or protected amino acid residues, covalently bound to the polymer directly or through side groups.
In the preferred embodiment, the bioactive substituents are linked to the polymer via the amino groups on the amino acid moieties directly or via a suitable spacer molecule. The spacer molecule can be selected from xcex1-hydroxy acids represented by the formula R7R8COHCO2H, where R7 or R8 groups are independently selected from H, linear or branched alkyl units and xcex1-amino acids represented by the formula R9CHNHR10CO2H, where the R9 group is a hydroxyl methyl or methyl thiol group and R10 is an amine protecting group.
In accordance with a further aspect of the invention, the invention provides a method of making a biodegradable, biocompatible polyester, which comprises co-polymerizing at least one xcex1-hydroxy acid and at least one pseudo-xcex1-amino acid.
In accordance with another aspect of the present invention, there is provided a process for making a biodegradable, biocompatible polymer of the general formula provided herein which comprises forming a mixture of monomers comprising at least one xcex1-hydroxy acid and at least one pseudo-xcex1-amino acid with an organic solvent solution of an esterification catalyst under inert atmospheric conditions, copolymerizing the monomers and isolating the resultant polymer. The catalyst used is preferably stannous 2-ethylhexanoate.
The polymer formed by the process can be further deprotected by solid phase catalytic reduction or alternatively by acid catalysis using hydrogen bromide in acetic acid solution.
The process can also further comprise forming the polymer into a film or microparticles.
In accordance with another aspect of this invention, there is provided a particulate carrier for the delivery of biologically active materials to a host, the carrier comprising a polymer, including those having a molecular weight of about 5,000 to about 40,000 daltons, having the general formula: 
wherein;
R1, R2, R3, R4 and R5 are selected independently and are selected from H, linear or branched alkyl groups;
R6 is selected from H, an amine protecting group, a spacer molecule or a biologically active species;
X is selected from an O or S group; and
x and y are integers, including values such that at least about 95% of the polymer is comprised of xcex1-hydroxy acid residues.
The particulate carrier generally has a particle size of about 1 to 50 xcexcM.
In a further aspect of the present invention is a process for making a particulate carrier for the delivery of at least one biologically active material to a host, the process comprising:
(a) mixing an organic solvent phase comprising an xcex1-hydroxy acid polymer or copolymer with an aqueous composition comprising dispersed or dissolved biologically active material to form a first water-in-oil emulsion;
(b) dispersing the first water-in-oil emulsion into an aqueous detergent phase to form a second water-in-oil-in-water double emulsion;
(C) removing water from the second double emulsion to form microspheres; and
(d) collecting the microspheres and having the biological material entrapped therein.
The particulate carrier of the present invention can be used as a composition having a biologically active material mixed therewith or entrapped within. The biological materials used may be selected from those which elicit an immune response. Such materials may comprise Haemophilus influenzae proteins, such as a non-proteolytic Hin-47 analog, D15, P1, P2, and P6. The biologically-active material may comprise at least one influenza virus, which may be a multivalent or monovalent influenza virus vaccine, or influenza virus protein, such as an influenza virus monovalent protein vaccine. In addition, the biologically-active material may comprise at least one Moraxella catarrhalis protein, such as the Tbp2 protein of M. catarrhalis. A further biologically-active material which may be employed may be at least one Helicobacter pylori protein, such as Urease. Other biological material may include proteins, protein mimetics, bacteria, bacterial lysates, viruses (e.g. respiratory syncytial virus), virus-infected cell lysates, DNA plasmids, antisense RNA, peptides (e.g. CLTB-36 and M2), antigens, antibodies, pharmacological agents, antibiotics, carbohydrates, lipids, lipidated amino acids (e.g. tripalmitoyl cysteine), glycolipids, haptens and combinations and mixtures thereof.
The first water-in-oil emulsion may additionally comprise at least one organic solvent soluble adjuvant, which may be lipophilic. Such organic solvent adjuvant may be selected from the group consisting of BAY R1-005, tripalmitoyl cysteine and DC-chol. The presence of a lipophilic moiety serves to increase the encapsulation efficiency and to protect the antigen during formulation and release and enables the particles to present antigen to the immune system more efficiently than traditional formulation and hence provides a more efficacious vaccine.
The first water-in-oil emulsion also may additionally comprise at least one water soluble adjuvant, which may be a polymeric water soluble adjuvant, such as PCPP or a mucosal adjuvant, such as CT-X or subunit thereof or LT. The presence of the water soluble adjuvant serves to increase the encapsulation efficiency of the process and protects the antigen during formulation and release and prevents the antigen to the immune system more efficiently than traditional microparticle formulation, thereby providing a more efficancious vaccine.
The present invention also provides an immunogenic composition comprising the particulate carrier provided herein and a physiologically acceptable carrier therefor. The composition can be administered mucosally or parenterally. The immune response is an antibody response which is a local or serum antibody response. In accordance with this aspect of the invention, there is provided a controlled or delayed release vaccine preparation in stable particulate form and a method of making such a vaccine preparation. The particles are microspherical and contain a matrix of biodegradable polymer and antigen(s) and/or antigen plus co-adjuvant containing regions.
Advantages of the invention include:
(a) fully biodegradable and biocompatible microparticle formulation;
(b) facilitated antigen presentation to the cells of the immune system resulting in improved antigen immunogenicity;
(c) improved formulating conditions which increase the bioavailability of the antigen.
Additional embodiments of the present invention include the use of the particulate carrier in diagnostic assays and for therapeutic strategies.